Materials and methods for synthesis of a flavor and aroma volatile in plants

ABSTRACT

The subject invention concerns polynucleotides encoding a plant 2-phenylethanol dehydrogenase enzyme. In one embodiment, the polynucleotide encodes a tomato 2-phenylethanol dehydrogenase. The subject invention also concerns polynucleotides encoding a plant phenylalanine decarboxylase enzyme. In one embodiment, the polynucleotide encodes a tomato phenylalanine decarboxylase. The subject invention also concerns 2-phenylethanol dehydrogenase polypeptides and phenylalanine decarboxylase polypeptides encoded by polynucleotides of the present invention. The subject invention also concerns methods for providing a plant with an increased flavor and aroma volatile. Plants can be transformed with one or more polynucleotide of the present invention. The subject invention also concerns these transformed plant cells, plant tissue, and plants and transgenic progeny thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. national stage application of International patent application No. PCT/US2004/032599, filed Oct. 1, 2004, which claims the benefit of U.S. Provisional Application Ser. No. 60/508,568, filed Oct. 3, 2003 and U.S. Provisional Application Ser. No. 60/558,504, filed Mar. 31, 2004.

This invention was made with government support under National Science Foundation grant number 7223426-12. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Fruits are major components of the human diet contributing a large portion of vitamins, minerals, antioxidants, and fiber. While flavor and nutrition composition have clear and profound potential for positive human benefit, they have proven to be difficult traits to modify via either traditional breeding or transgenic approaches due to their generally complex biosynthetic and regulatory pathways. In fact, the biochemical descriptors that comprise flavor are poorly defined. What is typically perceived as flavor in many fruits is the product of a complex interaction among sugars, acids and multiple volatile secondary metabolites (Buttery et al., 1988; Baldwin et al., 2000). Synthesis and accumulation of these compounds is the result of coordinated activity of many genes that may also impact additional aspects of plant growth and development. Effective manipulation of these traits for human benefit will therefore require greater knowledge of the pathways involved and the regulatory systems which control them. Prior to the advent of genomics, researchers could focus on the activity of only one to several genes important in a process of interest and could view their respective effects in relative isolation. From a practical perspective, flavor and nutrition are intimately related and equally important as flavor directly impacts the choice of foods for consumption which, in turn, has positive nutritional consequences on the human diet.

Fruit-bearing crop plants are taxonomically diverse (e.g., pepper, tomato, melons, apples, bananas, grapes). However, they do share common features; most, though not all, fruits are enlarged ovaries. While our knowledge of how domesticated plants came to bear fruit or the mechanisms by which they ripen is still rudimentary, more is known about these processes in tomato (Lycopersicon esculentum) than in any other species (see Giovannoni (2001) for review). Furthermore, a diverse set of Near Isogenic Lines (NILs), single gene ripening mutants, and transgenic lines represent portals through which genetic regulation of fruit development and ripening can be studied (Gray et al., 1994; Giovannoni et al., 1999). The diversity of genetically well characterized tomato germplasm described below (Table 1) is unparalleled in other fruiting species. Nevertheless, it is important to realize that while fruit ripening is a complex sum of coordinately regulated biochemical events that vary from species to species, key regulatory components are likely to be maintained (Hobson and Grierson, 1993). For example, one group has recently identified two genes that are essential for fruit ripening, RIN and NOR (Giovannoni, 2001; Vrebalov et al., 2002). Fruit-specific, ripening-induced homologues of these genes have been identified from strawberry and banana (Vrebalov et al., 2002). Strawberry undergoes a very different ripening program as compared to tomato in that strawberry is non-climacteric (i.e. no increase in respiration or ethylene biosynthesis during ripening) and accumulates high levels of anthocyanins rather than carotenoids during fruit maturation. Further, it is anatomically a receptacle, whereas most fruits are ovaries. Banana is interesting in that while its fruit are also expanded carpels, it is a monocot. Apparently similar ripening control shared among monocots and dicots indicates that basic ripening regulation is likely conserved through evolution. In summary, these results suggest that while specific nutritional and flavor components may vary among fruit species they are likely due to regulated metabolic flux through similar pathways with similar genetic control systems. Thus, regulatory and biosynthetic genes identified in tomato will allow for modification of the same or related compounds in a wide range of agriculturally important fruit species.

Tomato has long served as a model system for plant genetics, development, physiology and fruit ripening resulting in the accumulation of substantial information regarding the biology of this economically important plant. Many experimental tools and features of tomato make it ideal for study of fruit ripening; these include extensive germplasm collections, numerous natural, induced, and transgenic mutants, routine transformation technology, a dense and expanding RFLP map, numerous cDNA and genomic libraries, a small genome, relatively short life-cycle and ease of growth and maintenance. In addition, numerous genomic tools that have and continue to be developed include: a) over 140,000 EST sequences (˜30,000 non-redundant) from 23 different tomato tissues/treatments (with one-third of the ESTs derived from fruit), b) EST expression arrays being developed and utilized (see bti.cornell.edu/CGEP/CGEP.html) and c) recent initiation of activities toward development of a tomato physical map anchored to the genetic map to facilitate gene isolation and eventual genome sequencing (Tanksley et al., NSF tomato genome project, 1992). The intense research effort in tomato fruit biology has resulted in many important discoveries that have had a broad impact on the field of plant biology, including control of gene expression by antisense technology, characterization of numerous genes influencing fruit development and ripening, characterization of genes for ethylene synthesis and perception, and the recent connection of ripening regulation and ethylene response to the molecular regulation of floral development (Vrebalov et al., 2002).

Fruit maturation and ripening is the summation of biochemical and physiological changes occurring at the terminal stage of development rendering the organ edible and valuable as an agricultural commodity. These changes frequently include modification of cell wall ultrastructure and texture, conversion of starch to sugars, alterations in pigment and nutrient biosynthesis/accumulation, and heightened levels of flavor and aromatic volatiles (Rhodes, 1980; Hobson and Grierson, 1993). While some ripening effects, such as carotenoid and vitamin C synthesis and accumulation, have direct impact on the nutritive value of mature fruit, others impacting flavor and texture (e.g., volatiles, sugars and acids) can have an indirect impact on human nutrition via their contributions to total consumption levels. In short, “if it tastes better” consumption will increase. This is especially critical as poor food choices exert a disproportional impact on children and members of society on lower rungs of the socio-economic ladder.

Although most fruits display modifications in color, texture, flavor and nutrient composition during maturation, two major classifications of ripening, climacteric and non-climacteric, have been utilized to distinguish fruit on the basis of respiration and ethylene synthesis rates. Climacteric fruits such as tomato, avocado, banana, peaches and apples, are distinguished from non-climacteric fruits such as strawberry, grape and citrus, by their increased respiration and ethylene synthesis rates during ripening (Lelievre et al., 1998). In tomato, ethylene has been shown to be necessary for the coordination and completion of ripening (Yang, 1985; Tucker and Brady, 1987; Klee et al., 1991; Picton et al., 1993; Lanahan et al., 1994). The critical role of ethylene in coordinating climacteric ripening at the molecular level was first observed via analysis of ethylene inducible ripening-related gene expression in tomato (Lincoln et al., 1987; Maunders et al., 1987). Numerous fruit development-related genes have since been isolated via differential expression patterns and biochemical function (reviewed in Gray et al., 1994). The in vivo functions of many fruit development- and ripening-related genes have been tested via antisense repression and/or mutant complementation in tomato. As examples, polygalacturonase was shown to be necessary for ripening-related pectin depolymerization and pathogen susceptibility, yet to have little effect on fruit softening (Smith et al., 1988, Giovannoni et al., 1989, Kramer et al., 1990). Inhibition of phytoene synthase resulted in reduced carotenoid biosynthesis and reduction in fruit and flower pigmentation (Fray and Grierson, 1993). Reduced ethylene evolution resulted in ripening inhibition of ACC synthase (ACS) and ACC oxidase (ACO) antisense lines (Oeller et al., 1991; Hamilton et al., 1990) while introduction of a dominant mutant allele of the NR ethylene receptor resulted in plants inhibited in virtually every measurable ethylene response including fruit ripening (Wilkinson et al., 1995; Yen et al., 1995).

Expression analysis of multiple tomato ripening-related genes indicates that a subset exhibit developmentally-controlled ethylene inducibility, i.e., they are ethylene inducible only in ripening fruits. Examples include members of the ACO and ACS gene families (Theologis et al., 1993; Blume and Grierson, 1997; Nakatsuka et al., 1998), the NR ethylene receptor (Wilkinson et al., 1995; Payton et al., 1996; Lashbrook et al., 1998) and E8 (Deikman et al., 1992). Additional evidence for non-ethylene mediated ripening control comes from analysis of gene expression in ripening impaired mutants such as rin (ripening-inhibitor) and nor (non-ripening) that fail to ripen in response to exogenous ethylene yet display signs of ethylene sensitivity and signaling including induction of some ethylene-regulated genes (Yen et al., 1995). These results suggest that regulatory constraints are placed on climacteric fruit maturation in addition to general ethylene biosynthesis and signaling. Such mechanisms could include fruit-specific regulation of certain subsets of ethylene regulated genes or factors that operate separate from and in addition to ethylene as seems to be the case for both the RIN (Vrebalov et al., 2002) and NOR transcription factors. This is particularly interesting as a greater understanding of the relationship between ethylene, developmental, and environmental signals will likely reveal the impact of various signaling systems on pathways impacting flavor and human nutrition. Indeed numerous environmental factors such as light and temperature can dramatically influence the degree and rate of fruit ripening with significant impacts on the accumulation of carotenoids and flavor compounds (Hobson and Grierson, 1993; Yen et al., 1997).

Numerous plant metabolites can be listed when the net of “nutritive compounds” is cast. These include various antioxidants, vitamins, minerals, fiber, lipids, and amino acids, to name just a few. In addition, as noted above, one can rationally argue that modification of flavor and additional quality attributes may lead to improved health via increased fruit or vegetable consumption.

Tomato fruits are among the highest source of lycopene, β-carotene, and vitamin C (ascorbate) in the diets of humans in the US, South America, and Europe, with steadily increasing prominence in Asia and the Middle East. In addition to direct nutritive value, carotenoids in particular are metabolized to compounds that impact flavor and aroma of fruit and thus have a significant impact on resulting fresh and processed products. Genes encoding the synthetic steps from phytoene through β-carotene (Bartley et al., 1994; Ronen et al., 1999) are potential regulatory points for modification of carotenoid levels. Indeed, available data indicate that accumulation of lycopene is due to coordinated up-regulation of the genes preceding its synthesis and down-regulation of genes that further metabolize it during ripening (Ronen et al., 1999). Numerous mutant, transgenic, RI and breeding lines that display a wide range of levels of lycopene and β-carotene are available (Table 1). While specific mutants represent some of the catalytic steps (e.g., r=phytoene synthase and cr and B=lycopene cyclase; Hamilton et al., 1990; Ronen et al., 1999) others such as hp-1 and hp-2 represent regulators of environmental response. Antisense phytoene synthase tomato lines are greatly reduced in all of the carotenoid-derived volatiles (Baldwin et al., 2000). Furthermore, transgenic and mutant lines altered in ethylene synthesis or perception display variation in carotenoid levels (Table 1).

TABLE 1 Tomato germplasm altered in carotenoids, flavonoids, vitamin C. Carotenoids Vit. C Volatiles Function Genotype rin; ripening-inhibitor* very low low NA MADS-box protein nor; non-ripening* low low NA transcription factor Nr: Never-ripe* low NA NA ethylene receptor hp-2; high-pigment-2 high high NA DET1 (light signaling) cr; crimson low B, high L high NA lycopene cyclase B; Beta high B, low L NA NA lycopene cyclase r; Phytoene Synthase low NA low phytoene synthase hp-1; high-pigment-1** high high NA Not cloned (light signaling) Nr-2: Never-ripe-2 low NA NA Not cloned Gr: Green-ripe low NA NA Not cloned t; tangerine low NA NA Not cloned at; apricot low NA NA Not cloned Cnr; Clear non-ripening low NA NA Not cloned L. esculentum × low-high low-high low-high L. pennellii Recombinant Inbreds ACO; ACC oxidase* low NA NA ethylene Biosynthesis ACS; ACC synthase* low NA NA ethylene Biosynthesis ACD; ACC deaminase* low NA NA ethylene Biosynthesis TCTR1; tomato CTR1* low-high NA NA ethylene signaling MAPKKK The dashed line separates mutants for which the corresponding gene has been cloned (1st tier) from those which have not (2nd tier). The last tier indicates transgenic lines altered in ethylene synthesis or response and with corresponding changes in carotenoid accumulation. Genotypes indicated with an (*) represent those for which multiple independent transgenic lines are available demonstrating a range of carotenoid accumulation levels. **Three different mutant alleles of hp-1 each having varying degrees of effect on carotenoid and flavonoid accumulation were provided by M. Koornneef. B = β-carotene. L = lycopene. While quantitative data for vitamin C and volatiles are unknown for many of these lines (NA), their respective phenotypes suggest they are likely to be altered in one or both.

In the case of flavor volatiles, the pathways for synthesis are in many cases not well established. For example, synthesis of apocarotenoids such as β-ionone and β-damascenone is not at all understood. Only recently has an Arabidopsis enzyme, CCD1 (Carotenoid Cleavage Dioxygenase), that synthesizes apocarotenoids such as β-ionone in vitro been identified (Schwartz et al., 2001). This gene is part of a multigene family, some of which are responsible for synthesis of other apocarotenoids such as ABA (Tan et al., 1997). CCD 1 cleaves multiple carotenoid substrates at the 9-10 and 9′-10′ bonds, potentially releasing volatiles such as β-ionone, although this has not been established in vivo. Similarly, several different volatiles are derived from lipid breakdown (Table 2). The likely first step in their syntheses is the action of a lipoxygenase (LOX) (Riley and Thompson, 1997; Baldwin et al., 2000). Currently there are 14 different EST contigs in the tomato database putatively identified as LOX. Any LOX exhibiting correlation with the lipid-derived volatiles would be a candidate sequence for analyses. It is exactly this sort of correlative biochemical and expression approach that resulted in identification of a key enzyme in strawberry volatile synthesis (Aharoni et al., 2000).

TABLE 2 The 16 most significant flavor volatiles of tomato Conc. Log odor Odor Volatile (ppb) units Precursor Characteristics cis-3-Hexenal 12,000 3.7 lipid Tomato/green β-ionone 4 2.8 carotenoid fruity/floral Hexanal 3,100 2.8 lipid green/grassy β-Damascenone 1 2.7 carotenoid Fruity 1-Penten-3-one 520 2.7 lipid fruity floral/green 2 + 3-Methylbutanal 27 2.1 ILE/LEU Musty trans-2-Hexenal 270 1.2 lipid Green 2-Isobutylthiazole 36 1.0 LEU Tomato vine 1-nitro-2-Phenylethane 17 0.9 PHE musty, earthy trans-2-Heptenal 60 0.7 lipid Green Phenylacetaldehyde 15 0.6 PHE floral/alcohol 6-Methyl-5-hepten- 130 0.4 carotenoid fruity, floral 2-one cis-3-Hexenol 150 0.3 lipid Green 2-Phenylethanol 1,900 0.3 PHE Nutty 3-Methylbutanol 380 0.2 LEU earthy, musty Methyl salicylate 48 0.08 PHE wintergreen Volatiles are ranked by importance based on Odor Units (concentration X humans' ability to detect). Concentrations are average values from typical commercial tomatoes. Odor characteristics were determined by a trained expert panel.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns polynucleotides encoding a plant 2-phenylethanol dehydrogenase enzyme. In one embodiment, a polynucleotide encodes a tomato 2-phenylethanol dehydrogenase. In another embodiment, a polynucleotide encodes a petunia 2-phenylethanol dehydrogenase. The subject invention also concerns 2-phenylethanol dehydrogenase polypeptides encoded by polynucleotides of the present invention.

The subject invention concerns polynucleotides encoding a plant phenylalanine decarboxylase enzyme. In one embodiment, a polynucleotide encodes a tomato phenylalanine decarboxylase. The subject invention also concerns phenylalanine decarboxylase polypeptides encoded by polynucleotides of the present invention.

The subject invention also concerns methods for providing a plant with increased flavor and aroma volatiles. Plants can be transformed with one or more polynucleotides of the present invention. The subject invention also concerns these transformed plant cells, plant tissues, and plants and transgenic progeny thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show the results of GC profiles of volatiles collected from M82 or introgression line 8-2-1 ripe fruit. Introgression line 8-2-1 fruit have higher levels of phenylacetaldehyde and 2-phenylethanol than control M82 fruit.

FIG. 2 is a graph of phenylacetaldehyde and 2-phenylethanol levels in control M82 and L. pennellii introgression line 8-2-1 fruit. Data are presented as % of control M82 fruit.

FIGS. 3A and 3B show a full-length cDNA (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of a 2-phenylethanol dehydrogenase of the present invention.

FIG. 4 shows a pathway for production of the volatiles phenylacetaldehyde and 2-phenylethanol in plants. Phenylalanine is decarboxylated by phenylalanine decarboxylase to form phenethylamine. Phenethylamine is then converted to phenylacetaldehyde by an amine oxidase, followed by conversion to 2-phenylethanol by 2-phenylethanol dehydrogenase.

FIGS. 5A, 5B, and 5C show the result of alcohol dehydrogenase activities of 2-phenylethanol dehydrogenase on phenylacetaldehyde and related substrates. Activity is determined by the disappearance of substrate and a reduction in OD (340 nm). Highest activity levels are observed with phenylacetaldehyde as a substrate.

FIGS. 6A and 6B show the result of GC profiles of volatiles emitted from wild-type Mitchell Diploid (MD) petunia flowers and transgenic petunia flowers expressing a tomato 2-phenylethanol dehydrogenase gene. In transgenic flowers, higher levels of 2-phenylethanol and lower levels of phenylacetaldehyde are observed as compared to wild-type flowers.

FIG. 7 is a graph of the levels of 2-phenylethanol and phenylacetaldehyde emitted from petunia flowers of wild-type Mitchell Diploid (MD) and transgenic lines expressing a tomato 2-phenylethanol dehydrogenase gene.

FIGS. 8A and 8B show a full-length coding sequence (SEQ ID NO: 4) and amino acid sequence (SEQ ID NO: 5) of tomato phenylalanine decarboxylase cLEC75E21 of the present invention.

FIGS. 9A and 9B show a full-length coding sequence (SEQ ID NO: 6) and amino acid sequence (SEQ ID NO: 7) of tomato phenylalanine decarboxylase cLEC73K23 of the present invention.

FIG. 10 is a Southern blot showing that cLEC73K23 is present on the L. pennellii introgression line (IL) 8-2-1 on tomato chromosome 8. Restriction patterns of L. pennellii and IL8-2-1 genomic DNA hybridized to the cLEC73K23 cDNA are identical, whereas L. esculentum M82 restriction patterns are different.

FIGS. 11A and 11B show a full-length coding sequence (SEQ ID NO: 8) and amino acid sequence (SEQ ID NO: 9) of an L. pennellii phenylalanine decarboxylase similar to cLEC73K23 of the present invention.

FIGS. 12A and 12B show an amino acid sequence alignment of L. esculentum cLEC73K23 (SEQ ID NO: 7), L. esculentum cLEC75E21 (SEQ ID NO: 5) and the L. pennellii cLEC73K23 homolog (SEQ ID NO: 9).

FIGS. 13A, 13B, and 13C show gas chromatography profiles of volatile compounds extracted from E. coli cultures expressing the tomato aromatic amino acid decarboxylase genes cLEC73K23 and cLEC75E21. E. coli expressing these tomato genes produce phenethylamine in media containing phenylalanine, while control E. coli cultures do not.

FIGS. 14A-14B show the nucleotide (SEQ ID NO: 10) and amino acid sequence (SEQ ID NO: 11) of a petunia homolog of the tomato phenylethanol dehydrogenase. The E. coli expressed protein exhibits phenylethanol dehydrogenase activity in vitro.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 shows a nucleotide sequence encoding a 2-phenylethanol dehydrogenase according to the present invention.

SEQ ID NO: 2 shows an amino acid sequence of a 2-phenylethanol dehydrogenase encoded by SEQ ID NO: 1 of the present invention.

SEQ ID NO: 3 shows an oligonucleotide PCR primer that can be used according to the present invention.

SEQ ID NO: 4 shows a nucleotide sequence encoding a phenylalanine decarboxylase according to the present invention.

SEQ ID NO: 5 shows an amino acid sequence of a phenylalanine decarboxylase encoded by SEQ ID NO: 4 of the present invention.

SEQ ID NO: 6 shows a nucleotide sequence encoding a phenylalanine decarboxylase according to the present invention.

SEQ ID NO: 7 shows an amino acid sequence of a phenylalanine decarboxylase encoded by SEQ ID NO: 6 of the present invention.

SEQ ID NO: 8 shows a nucleotide sequence encoding a phenylalanine decarboxylase according to the present invention.

SEQ ID NO: 9 shows an amino acid sequence of a phenylalanine decarboxylase encoded by SEQ ID NO: 8 of the present invention.

SEQ ID NO: 10 shows a nucleotide sequence encoding a petunia homolog of a tomato 2-phenylethanol dehydrogenase according to the present invention.

SEQ ID NO: 11 shows an amino acid sequence of a 2-phenylethanol dehydrogenase encoded by SEQ ID NO: 10 of the present invention.

SEQ ID NO: 12 shows a nucleotide sequence encoding a phenylalanine decarboxylase according to the present invention.

SEQ ID NO: 13 shows an amino acid sequence of a phenylalanine decarboxylase encoded by SEQ ID NO: 12 of the present invention.

DETAILED DISCLOSURE OF THE INVENTION

The subject invention concerns polynucleotides encoding a plant 2-phenylethanol dehydrogenase enzyme. In one embodiment, the polynucleotide encodes a 2-phenylethanol dehydrogenase of tomato; however, polynucleotides encoding homologous 2-phenylethanol dehydrogenase polypeptides of other plant species are also contemplated within the scope of the present invention. In another embodiment, the polynucleotide encodes a 2-phenylethanol dehydrogenase of petunia. In an exemplified embodiment, the polynucleotide encodes a tomato 2-phenylethanol dehydrogenase polypeptide having an amino acid sequence shown in SEQ ID NO: 2, or an enzymatically active fragment or variant thereof. In another exemplified embodiment, the polynucleotide encodes a petunia 2-phenylethanol dehydrogenase polypeptide having an amino acid sequence shown in SEQ ID NO: 11, or an enzymatically active fragment or variant thereof. In a specific embodiment, the polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 2 comprises the nucleotide sequence shown in SEQ ID NO: 1, or a sequence encoding an enzymatically active fragment or variant of SEQ ID NO: 2. In a further specific embodiment, the polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 11 comprises the nucleotide sequence shown in SEQ ID NO: 10, or a sequence encoding an enzymatically active fragment or variant of SEQ ID NO: 11. Thus, the subject invention concerns polynucleotide sequences comprising the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 11, or a variant, including a degenerate variant, of SEQ ID NO: 1 or SEQ ID NO: 11. In one embodiment, the polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 2 comprises nucleotides 172 to 1155 of the nucleotide sequence shown in SEQ ID NO: 1. In one embodiment, the polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 11 comprises nucleotides 1 to 990 of the nucleotide sequence shown in SEQ ID NO: 10.

The subject invention concerns polynucleotides encoding a plant phenylalanine decarboxylase enzyme. In one embodiment, the polynucleotide encodes a phenylalanine decarboxylase of tomato; however, polynucleotides encoding homologous phenylalanine decarboxylase polypeptides of other plant species are also contemplated within the scope of the present invention. In an exemplified embodiment, the polynucleotide encodes a tomato phenylalanine decarboxylase polypeptide having an amino acid sequence shown in SEQ ID NO: 5, 7, 9, or 13, or an enzymatically active fragment or variant thereof. In a specific embodiment, the polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 5, 7, 9, or 13 comprises the nucleotide sequence shown in SEQ ID NO: 4, 6, 8, or 12, respectively, or a sequence encoding an enzymatically active fragment or variant of SEQ ID NO: 5, 7, 9, or 13. Thus, the subject invention concerns polynucleotide sequences comprising the nucleotide sequence shown in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 12, or a variant, including a degenerate variant, of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 12. In one embodiment, the polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 5 comprises nucleotides 1 to 1395 of the nucleotide sequence shown in SEQ ID NO: 4. In another embodiment, the polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 7, 9, or 13 comprises nucleotides 1 to 1413 of the nucleotide sequence shown in SEQ ID NO: 6, 8, or 12, respectively.

The subject invention also concerns polynucleotide expression constructs comprising a polynucleotide sequence of the present invention encoding a plant 2-phenylethanol dehydrogenase. In one embodiment, an expression construct of the invention comprises a polynucleotide sequence encoding a tomato 2-phenylethanol dehydrogenase comprising an amino acid sequence shown in SEQ ID NO: 2, or an enzymatically active fragment or variant thereof. In another exemplified embodiment, an expression construct of the invention comprises a polynucleotide sequence encoding a petunia 2-phenylethanol dehydrogenase polypeptide having an amino acid sequence shown in SEQ ID NO: 11, or an enzymatically active fragment or variant thereof. In a specific embodiment, the polynucleotide sequence comprises a polynucleotide sequence shown in SEQ ID NO: 1, or a sequence encoding an enzymatically active fragment or variant of SEQ ID NO: 2. In a further specific embodiment, the polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 11 comprises the nucleotide sequence shown in SEQ ID NO: 10, or a sequence encoding an enzymatically active fragment or variant of SEQ ID NO: 11. In one embodiment, the polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 2 comprises nucleotides 172 to 1155 of the nucleotide sequence shown in SEQ ID NO: 1. In one embodiment, the polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 11 comprises nucleotides 1 to 990 of the nucleotide sequence shown in SEQ ID NO: 10.

The subject invention also concerns polynucleotide expression constructs comprising a polynucleotide sequence of the present invention encoding a plant phenylalanine decarboxylase. In one embodiment, an expression construct of the invention comprises a polynucleotide sequence encoding a tomato phenylalanine decarboxylase comprising an amino acid sequence shown in SEQ ID NO: 5, 7, 9, or 13, or an enzymatically active fragment or variant thereof. In a specific embodiment, the polynucleotide sequence comprises a polynucleotide sequence shown in SEQ ID NO: 4, 6, 8, or 12, or a sequence encoding an enzymatically active fragment or variant of SEQ ID NO: 5, 7, 9, or 13. In one embodiment, the polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 5 comprises nucleotides 1 to 1395 of the nucleotide sequence shown in SEQ ID NO: 4. In another embodiment, the polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 7, 9, or 13 comprises nucleotides 1 to 1413 of the nucleotide sequence shown in SEQ ID NO: 6, 8, or 12, respectively.

Expression constructs of the invention generally include regulatory elements that are functional in the intended host cell in which the expression construct is to be expressed. Thus, a person of ordinary skill in the art can select regulatory elements for use in bacterial host cells, yeast host cells, plant host cells, insect host cells, mammalian host cells, and human host cells. Regulatory elements include promoters, transcription termination sequences, translation termination sequences, enhancers, and polyadenylation elements. As used herein, the term “expression construct” refers to a combination of nucleic acid sequences that provides for transcription of an operably linked nucleic acid sequence. As used herein, the term “operably linked” refers to a juxtaposition of the components described wherein the components are in a relationship that permits them to function in their intended manner. In general, operably linked components are in contiguous relation.

An expression construct of the invention can comprise a promoter sequence operably linked to a polynucleotide sequence encoding a 2-phenylethanol dehydrogenase or phenylalanine decarboxylase of the invention. In one embodiment, the promoter is one that provides for overexpression of a polynucleotide of the present invention. Promoters useful for overexpression of an operably linked nucleic acid sequence are known in the art. Promoters can be incorporated into a polynucleotide sequence or an expression construct using standard techniques known in the art. Multiple copies of promoters or multiple promoters can be used in an expression construct of the invention. In a preferred embodiment, a promoter can be positioned about the same distance from the transcription start site in the expression construct as it is from the transcription start site in its natural genetic environment. Some variation in this distance is permitted without substantial decrease in promoter activity. A transcription start site is typically included in the expression construct.

If the expression construct is to be provided in or introduced into a plant cell, then plant viral promoters, such as, for example, a cauliflower mosaic virus (CaMV) 35S (including the enhanced CaMV 35S promoter (see, for example U.S. Pat. No. 5,106,739)) or a CaMV 19S promoter can be used. In an exemplified embodiment, the promoter is a figwort mosaic virus 35S promoter. Other promoters that can be used for expression constructs in plants include, for example, prolifera promoter, Ap3 promoter, heat shock promoters, T-DNA 1′- or 2′-promoter of A. tumefaciens, polygalacturonase promoter, chalcone synthase A (CHS-A) promoter from petunia, tobacco PR-1a promoter, ubiquitin promoter (U.S. Pat. Nos. 6,528,701 and 6,054,574), actin promoter, alcA gene promoter, pin2 promoter (Xu et al., 1993), maize WipI promoter, maize trpA gene promoter (U.S. Pat. No. 5,625,136), maize CDPK gene promoter, and RUBISCO SSU promoter (U.S. Pat. No. 5,034,322) can also be used. Tissue-specific promoters, for example fruit-specific promoters, such as the E8 promoter of tomato (accession number: AF515784; Good et al. (1994)), a hybrid E4/E8 promoter (U.S. Pat. No. 6,118,049), the LeExp-1 promoter (U.S. Pat. No. 6,340,748), and the polygalacturonase-β subunit promoter of tomato (U.S. Pat. No. 6,127,179) can be used. Flower organ-specific promoters can be used with an expression construct of the present invention for expressing a polynucleotide of the invention in the flower organ of a plant. Examples of flower organ-specific promoters include any of the promoter sequences described in U.S. Pat. Nos. 6,462,185; 5,859,328; 5,652,354; 5,639,948; and 5,589,610. Seed-specific promoters such as the promoter from a (1-phaseolin gene (e.g., of kidney bean) or a glycinin gene (e.g., of soybean), and others, can also be used. Root-specific promoters, such as any of the promoter sequences described in U.S. Pat. No. 6,455,760 or U.S. Pat. No. 6,696,623, or in published U.S. patent application Nos. 20040078841; 20040067506; 20040019934; 20030177536; 20030084486; or 20040123349, can be used with an expression construct of the invention. Constitutive promoters (such as an CaMV, ubiquitin, actin, or NOS promoter), developmentally-regulated promoters, and inducible promoters (such as those promoters than can be induced by heat, light, hormones, or chemicals) are also contemplated for use with polynucleotide expression constructs of the invention.

For expression in animal cells, an expression construct of the invention can comprise suitable promoters that can drive transcription of the polynucleotide sequence. If the cells are mammalian cells, then promoters such as, for example, actin promoter, metallothionein promoter, NF-kappaB promoter, EGR promoter, SRE promoter, IL-2 promoter, NFAT promoter, osteocalcin promoter, SV40 early promoter and SV40 late promoter, Lck promoter, BMP5 promoter, TRP-1 promoter, murine mammary tumor virus long terminal repeat promoter, STAT promoter, or an immunoglobulin promoter can be used in the expression construct. The baculovirus polyhedrin promoter can be used with an expression construct of the invention for expression in insect cells.

For expression in prokaryotic systems, an expression construct of the invention can comprise promoters such as, for example, alkaline phosphatase promoter, tryptophan (trp) promoter, lambda P_(L), promoter, β-lactamase promoter, lactose promoter, phoA promoter, T3 promoter, T7 promoter, or tac promoter (de Boer et al., 1983).

Promoters suitable for use with an expression construct of the invention in yeast cells include, but are not limited to, 3-phosphoglycerate kinase promoter, glyceraldehyde-3-phosphate dehydrogenase promoter, metallothionein promoter, alcohol dehydrogenase-2 promoter, and hexokinase promoter.

Expression constructs of the invention may optionally contain a transcription termination sequence, a translation termination sequence, a sequence encoding a signal peptide, and/or enhancer elements. Transcription termination regions can typically be obtained from the 3′ untranslated region of a eukaryotic or viral gene sequence. Transcription termination sequences can be positioned downstream of a coding sequence to provide for efficient termination. A signal peptide sequence is a short amino acid sequence typically present at the amino terminus of a protein that is responsible for the relocation of an operably linked mature polypeptide to a wide range of post-translational cellular destinations, ranging from a specific organelle compartment to sites of protein action and the extracellular environment. Targeting gene products to an intended cellular and/or extracellular destination through the use of an operably linked signal peptide sequence is contemplated for use with the polypeptides of the invention. Classical enhancers are cis-acting elements that increase gene transcription and can also be included in the expression construct. Classical enhancer elements are known in the art, and include, but are not limited to, the CaMV 35S enhancer element, cytomegalovirus (CMV) early promoter enhancer element, and the SV40 enhancer element. Intron-mediated enhancer elements that enhance gene expression are also known in the art. These elements must be present within the transcribed region and are orientation dependent. Examples include the maize shrunken-1 enhancer element (Clancy and Hannah, 2002).

DNA sequences which direct polyadenylation of mRNA transcribed from the expression construct can also be included in the expression construct, and include, but are not limited to, an octopine synthase or nopaline synthase signal. The expression constructs of the invention can also include a polynucleotide sequence that directs transposition of other genes, i.e., a transposon.

Expression constructs can also include one or more dominant selectable marker genes, including, for example, genes encoding antibiotic resistance and/or herbicide-resistance for selecting transformed cells. Antibiotic-resistance genes can provide for resistance to one or more of the following antibiotics: hygromycin, kanamycin, bleomycin, G418, streptomycin, paromomycin, neomycin, and spectinomycin. Kanamycin resistance can be provided by neomycin phosphotransferase (NPT II). Herbicide-resistance genes can provide for resistance to phosphinothricin acetyltransferase or glyphosate. Other markers used for cell transformation screening include genes encoding β-glucuronidase (GUS), β-galactosidase, luciferase, nopaline synthase, chloramphenicol acetyltransferase (CAT), green fluorescence protein (GFP), or enhanced GFP (Yang et al., 1996).

The subject invention also concerns polynucleotide vectors comprising a polynucleotide sequence of the invention that encodes a 2-phenylethanol dehydrogenase or phenylalanine decarboxylase of the invention. Unique restriction enzyme sites can be included at the 5′ and 3′ ends of an expression construct or polynucleotide of the invention to allow for insertion into a polynucleotide vector. As used herein, the term “vector” refers to any genetic element, including for example, plasmids, cosmids, chromosomes, phage, virus, and the like, which is capable of replication when associated with proper control elements and which can transfer polynucleotide sequences between cells. Vectors contain a nucleotide sequence that permits the vector to replicate in a selected host cell. A number of vectors are available for expression and/or cloning, and include, but are not limited to, pBR322, pUC series, M13 series, and pBLUESCRIPT vectors (Stratagene, La Jolla, Calif.).

The subject invention also concerns oligonucleotide probes and primers, such as polymerase chain reaction (PCR) primers, that can hybridize to a coding or non-coding sequence of a polynucleotide of the present invention. Oligonucleotide probes of the invention can be used in methods for detecting nucleic acid sequences encoding a 2-phenylethanol dehydrogenase or a phenylalanine decarboxylase. Oligonucleotide primers of the invention can be used in PCR methods and other methods involving nucleic acid amplification. In a preferred embodiment, a probe or primer of the invention can hybridize to a polynucleotide of the invention under stringent conditions. Probes and primers of the invention can optionally comprise a detectable label or reporter molecule, such as fluorescent molecules, enzymes, radioactive moiety (e.g., ³H, ³⁵S, ¹²⁵I, etc.), and the like. Probes and primers of the invention can be of any suitable length for the method or assay in which they are being employed. Typically, probes and primers of the invention will be 10 to 500 or more nucleotides in length. Probes and primers that are 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 81 to 90, 91 to 100 or more nucleotides in length are contemplated within the scope of the invention. Probes and primers of the invention can have complete (100%) nucleotide sequence identity with the polynucleotide sequence, or the sequence identity can be less than 100%. For example, sequence identity between a probe or primer and a sequence can be 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70% or any other percentage sequence identity so long as the probe or primer can hybridize under stringent conditions to a nucleotide sequence of a polynucleotide of the invention. Exemplified probes and primers of the invention include those having the nucleotide sequence of SEQ ID NO: 3, or a functional fragment or variant of SEQ ID NO. 3.

Polynucleotides of the present invention can be composed of either RNA or DNA. Preferably, the polynucleotides are composed of DNA. The subject invention also encompasses those polynucleotides that are complementary in sequence to the polynucleotides disclosed herein. Polynucleotides and polypeptides of the invention can be provided in purified or isolated form.

Because of the degeneracy of the genetic code, a variety of different polynucleotide sequences can encode 2-phenylethanol dehydrogenase and phenylalanine decarboxylase enzymes of the present invention. A table showing all possible triplet codons (and where U also stands for T) and the amino acid encoded by each codon is described in Lewin (1985). In addition, it is well within the skill of a person trained in the art to create alternative polynucleotide sequences encoding the same, or essentially the same, 2-phenylethanol dehydrogenase or phenylalanine decarboxylase enzymes of the subject invention. These degenerate variant or alternative polynucleotide sequences are within the scope of the subject invention. As used herein, references to “essentially the same” sequence refers to sequences which encode amino acid substitutions, deletions, additions, or insertions which do not materially alter the functional activity of the polypeptide encoded by the polynucleotides of the present invention. Allelic variants of the nucleotide sequences encoding a 2-phenylethanol dehydrogenase or phenylalanine decarboxylase of the invention are also encompassed within the scope of the invention.

The subject invention also concerns an isolated plant 2-phenylethanol dehydrogenase. In one embodiment, the 2-phenylethanol dehydrogenase is a 2-phenylethanol dehydrogenase of tomato. In another embodiment, the 2-phenylethanol dehydrogenase is a 2-phenylethanol dehydrogenase of petunia. In a specific embodiment, a 2-phenylethanol dehydrogenase has an amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 11, or an enzymatically active fragment or variant of SEQ ID NO: 2 or SEQ ID NO: 11. A 2-phenylethanol dehydrogenase enzyme of the invention can be purified using standard techniques known in the art. In one embodiment, a polynucleotide of the invention encoding a 2-phenylethanol dehydrogenase is incorporated into a microorganism, such as E. coli, and the 2-phenylethanol dehydrogenase expressed in the microorganism and then isolated therefrom.

The subject invention also concerns an isolated plant phenylalanine decarboxylase. In one embodiment, the phenylalanine decarboxylase is a phenylalanine decarboxylase of tomato. In a specific embodiment, a phenylalanine decarboxylase has an amino acid sequence as shown in SEQ ID NO: 5, 7, 9, or 13, or an enzymatically active fragment or variant thereof. A phenylalanine decarboxylase enzyme of the invention can be purified using standard techniques known in the art. In one embodiment, a polynucleotide of the invention encoding a phenylalanine decarboxylase is incorporated into a microorganism, such as E. coli, and the phenylalanine decarboxylase expressed in the microorganism and then isolated therefrom.

Polypeptides of the invention, and peptide fragments thereof, can be used to generate antibodies that bind specifically to a polypeptide of the invention, and such antibodies are contemplated within the scope of the invention. The antibodies of the invention can be polyclonal or monoclonal and can be produced and isolated using standard methods known in the art.

Polypeptide fragments according to the subject invention typically comprise a contiguous span of about or at least 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193; 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, or 327 amino acids of SEQ ID NO: 2; or a contiguous span of about or at least 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, or 464 amino acids of SEQ ID NO: 5; or a contiguous span of about or at least 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, or 470 amino acids of SEQ ID NO: 7, 9, or 13; or a contiguous span of about or at least 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, or 329 amino acids of SEQ ID NO: 11.

Polypeptide fragments of the subject invention can be any integer in length from at least about 25 consecutive amino acids to 1 amino acid less than the sequence shown in SEQ ID NO: 2, 5, 7, 9, 11, or 13. Thus, for SEQ ID NO: 2, a polypeptide fragment can be any integer of consecutive amino acids from about 25 to 327 amino acids; for SEQ ID NO: 5, a polypeptide fragment can be any integer of consecutive amino acids from about 25 to 464 amino acids; for SEQ ID NO: 7, 9, or 13, a polypeptide fragment can be any integer of consecutive amino acids from about 25 to 470 amino acids; for SEQ ID NO: 11, a polypeptide fragment can be any integer of consecutive amino acids from about 25 to 329 amino acids. The term “integer” is used herein in its mathematical sense and thus representative integers include: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, and/or 470.

Each polypeptide fragment of the subject invention can also be described in terms of its N-terminal and C-terminal positions. For example, combinations of N-terminal to C-terminal fragments of about 25 contiguous amino acids to 1 amino acid less than the full length polypeptide of SEQ ID NO: 2, 5, 7, 9, 11, and 13 are included in the present invention. Thus, using SEQ ID NO: 2 as an example, a 25 consecutive amino acid fragment could correspond to amino acids of SEQ ID NO: 2 selected from the group consisting of 1-25, 2-26, 3-27, 4-28, 5-29, 6-30, 7-31, 8-32, 9-33, 10-34, 11-35, 12-36, 13-37, 14-38, 15-39, 16-40, 17-41, 18-42, 19-43, 20-44, 21-45, 22-46, 23-47, 24-48, 25-49, 26-50, 27-51, 28-52, 29-53, 30-54, 31-55, 32-56, 33-57, 34-58, 35-59, 36-60, 37-61, 38-62, 39-63, 40-64, 41-65, 42-66, 43-67, 44-68, 45-69, 46-70, 47-71, 48-72, 49-73, 50-74, 51-75, 52-76, 53-77, 54-78, 55-79, 56-80, 57-81, 58-82, 59-83, 60-84, 61-85, 62-86, 63-87, 64-88, 65-89, 66-90, 67-91, 68-92, 69-93, 70-94, 71-95, 72-96, 73-97, 74-98, 75-99, 76-100, 77-101, 78-102, 79-103, 80-104, 81-105, 82-106, 83-107, 84-108, 85-109, 86-110, 87-111, 88, -112, 89-113, 90-114, 91-115, 92-116, 93-117, 94-118, 95-119, 96-120, 97-121, 98422, 99-123, 100-124, 101-125, 102-126, 103-127, 104-128, 105-129, 106-130, 107-131, 108-132, 109-133, 110-134, 111-135, 112-136, 113-137, 114-138, 115-139, 116-140, 117-141, 118-142, 119-143, 120-144, 121-145, 122-146, 123-147, 124-148, 125-149, 126-150, 127-151, 128-152, 129-153, 130-154, 131-155, 132-156, 133-157, 134-158, 135-159, 136-160, 137-161, 138-162, 139-163, 140-164, 141-165, 142-166, 143-167, 144-168, 145-169, 146-170, 147-171, 148-172, 149-173, 150-174, 151-175, 152-176, 153-177, 154-178, 155-179, 156-180, 157-181, 158-182, 159-183, 160-184, 161-185, 162-186, 163-187, 164-188, 165-189, 166-190, 167-191, 168-192, 169-193, 170-194, 171-195, 172-196, 173-197, 174-198, 175-199, 176-200, 177-201, 178-202, 179-203, 180-204, 181-205, 182-206, 183-207, 184-208, 185-209, 186-210, 187-211, 188-212, 189-213, 190-214, 191-215, 192-216, 193-217, 194-218, 195-219, 196-220, 197-221, 198-222, 199-223, 200-224, 201-225, 202-226, 203-227, 204-228, 205-229, 206-230, 207-231, 208-232, 209-233, 210-234, 211-235, 212-236, 213-237, 214-238, 215-239, 216-240, 217-241, 218-242, 219-243, 220-244, 221-245, 222-246, 223-247, 224-248, 225-249, 226-250, 227-251, 228-252, 229-253, 230-254, 231-255, 232-256, 233-257, 234-258, 235-259, 236-260, 237-261, 238-262, 239-263, 240-264, 241-265, 242-266, 243-267, 244-268, 245-269, 246-270, 247-271, 248-272, 249-273, 250-274, 251-275, 252-276, 253-277, 254-278, 255-279, 256-280, 257-281, 258-282, 259-283, 260-284, 261-285, 262-286, 263-287, 264-288, 265-289, 266-290, 267-291, 268-292, 269-293, 270-294, 271-295, 272-296, 273-297, 274-298, 275-299, 276-300, 277-301, 278-302, 279-303, 280-304, 281-305, 282-306, 283-307, 284-308, 285-309, 286-310, 287-311, 288-312, 289-313, 290-314, 291-315, 292-316, 293-317, 294-318, 295-319, 296-320, 297-321, 298-322, 299-323, 300-324, 301-325, 302-326, 303-327, and 304-328. Similarly, the amino acids corresponding to all other fragments of sizes between 26 consecutive amino acids and 327 consecutive amino acids of SEQ ID NO: 2 are included in the present invention and can also be immediately envisaged based on these examples. Therefore, additional examples, illustrating various fragments of the polypeptides of SEQ ID NO: 2 are not individually listed herein in order to avoid unnecessarily lengthening the specification. Fragment embodiments as described above are also contemplated for the polypeptides of SEQ ID NO: 5, 7, 9, 11, and 13 taking into account that the polypeptides are 465, 471, 471, 330, and 471 amino acids in length, respectively, and are not individually listed herein in order to avoid unnecessarily lengthening the specification.

Polypeptide fragments comprising:

a) 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, or 327 consecutive amino acids of SEQ ID NO: 2;

b) 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, and 464 consecutive amino acids of SEQ ID NO: 5;

c) 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, and 470 consecutive amino acids of SEQ ID NO: 7, 9, or 13; and

d) 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, and 329 consecutive amino acids of SEQ ID NO: 11 may alternatively be described by the formula “n to c” (inclusive), where “n” equals the N-terminal amino acid position and “c” equals the C-terminal amino acid position of the polypeptide. In this embodiment of the invention, “n” is an integer having a lower limit of 1 and an upper limit of the total number of amino acids of the full length polypeptide minus 24 (e.g., 328−24=304 for SEQ ID NO: 2). “c” is an integer between 25 and the number of amino acids of the full length polypeptide sequence (328 for SEQ ID NO: 2) and “n” is an integer smaller then “c” by at least 24. Therefore, for SEQ ID NO: 2, “n” is any integer selected from the list consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, and 304; and “c” is any integer selected from the group consisting of: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, and 328 provided that “n” is a value less than “c” by at least 24. Every combination of “n” and “c” positions are included as specific embodiments of polypeptide fragments of the invention. Fragment embodiments as described above are also contemplated for the polypeptides of SEQ ID NO: 5, 7, 9, 11, and 13 taking into account that the polypeptides are 465, 471, 471, 330, and 471 amino acids in length, respectively, and are not individually listed herein in order to avoid unnecessarily lengthening the specification. All ranges used to describe any polypeptide fragment embodiment of the present invention are inclusive unless specifically set forth otherwise.

Fragments of a plant 2-phenylethanol dehydrogenase or phenylalanine decarboxylase of the invention, as described herein, can be obtained by cleaving the polypeptides of the invention with a proteolytic enzyme (such as trypsin, chymotrypsin, or collagenase) or with a chemical reagent, such as cyanogen bromide (CNBr). Alternatively, polypeptide fragments can be generated in a highly acidic environment, for example at pH 2.5. Polypeptide fragments can also be prepared by chemical synthesis or using host cells transformed with an expression vector comprising a polynucleotide encoding a fragment of a 2-phenylethanol dehydrogenase or phenylalanine decarboxylase enzyme of the invention, for example, a 2-phenylethanol dehydrogenase that is a fragment of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 11, or a phenylalanine decarboxylase that is a fragment of the amino acid sequence shown in SEQ ID NO: 5, 7, 9, or 13.

Substitution of amino acids other than those specifically exemplified or naturally present in a plant 2-phenylethanol dehydrogenase or phenylalanine decarboxylase enzyme of the invention are also contemplated within the scope of the present invention. For example, non-natural amino acids can be substituted for the amino acids of 2-phenylethanol dehydrogenase or phenylalanine decarboxylase, so long as the 2-phenylethanol dehydrogenase or phenylalanine decarboxylase enzyme having the substituted amino acids retains substantially the same biological activity as the 2-phenylethanol dehydrogenase or phenylalanine decarboxylase in which amino acids have not been substituted. Examples of non-natural amino acids include, but are not limited to, ornithine, citrulline, hydroxyproline, homoserine, phenylglycine, taurine, iodotyrosine, 2,4-diaminobutyric acid, α-amino isobutyric acid, 4-aminobutyric acid, 2-amino butyric acid, γ-amino butyric acid, ε-amino hexanoic acid, 6-amino hexanoic acid, 2-amino isobutyric acid, 3-amino propionic acid, norleucine, norvaline, sarcosine, homocitrulline, cysteic acid, τ-butylglycine, τ-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as β-methyl amino acids, C-methyl amino acids, N-methyl amino acids, and amino acid analogues in general. Non-natural amino acids also include amino acids having derivatized side groups. Furthermore, any of the amino acids in the protein can be of the D (dextrorotary) form or L (levorotary) form. Allelic variants of a protein sequence of 2-phenylethanol dehydrogenase and phenylalanine decarboxylase enzymes of the present invention are also encompassed within the scope of the invention.

Amino acids can be generally categorized in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby an 2-phenylethanol dehydrogenase or phenylalanine decarboxylase enzyme of the present invention having an amino acid of one class is replaced with another amino acid of the same class fall within the scope of the subject invention so long as the 2-phenylethanol dehydrogenase or phenylalanine decarboxylase enzyme having the substitution still retains substantially the same biological activity as the 2-phenylethanol dehydrogenase or phenylalanine decarboxylase enzyme that does not have the substitution. Polynucleotides encoding a 2-phenylethanol dehydrogenase or phenylalanine decarboxylase enzyme having one or more amino acid substitutions in the sequence are contemplated within the scope of the present invention. Table 3 below provides a listing of examples of amino acids belonging to each class.

TABLE 3 Class of Amino Acid Examples of Amino Acids Nonpolar Ala, Val, Leu, Ile, Pro, Met, Phe, Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr, Asn, Gln Acidic Asp, Glu Basic Lys, Arg, His

Single letter amino acid abbreviations are defined in Table 4.

TABLE 4 Letter Symbol Amino Acid A Alanine B Asparagine or aspartic acid C Cysteine D Aspartic Acid E Glutamic Acid F Phenylalanine G Glycine H Histidine I Isoleucine K Lysine L Leucine M Methionine N Asparagine P Proline Q Glutamine R Arginine S Serine T Threonine V Valine W Tryptophan Y Tyrosine Z Glutamine or glutamic acid

The subject invention also concerns variants of the polynucleotides of the present invention that encode enzymatically active 2-phenylethanol dehydrogenase or phenylalanine decarboxylase enzymes of the invention. Variant sequences include those sequences wherein one or more nucleotides of the sequence have been substituted, deleted, and/or inserted. The nucleotides that can be substituted for natural nucleotides of DNA have a base moiety that can include, but is not limited to, inosine, 5-fluorouracil, 5-bromouracil, hypoxanthine, 1-methylguanine, 5-methylcytosine, and tritylated bases. The sugar moiety of the nucleotide in a sequence can also be modified and includes, but is not limited to, arabinose, xylulose, and hexose. In addition, the adenine, cytosine, guanine, thymine, and uracil bases of the nucleotides can be modified with acetyl, methyl, and/or thio groups. Sequences containing nucleotide substitutions, deletions, and/or insertions can be prepared and tested using standard techniques known in the art.

Fragments and variants of 2-phenylethanol dehydrogenase and phenylalanine decarboxylase of the present invention can be generated as described herein and tested for the presence of enzymatic function using standard techniques known in the art. For example, for testing fragments and/or variants of a 2-phenylethanol dehydrogenase, the conversion of phenylacetaldehyde to 2-phenylethanol can be assayed according to the present invention. Thus, an ordinarily skilled artisan can readily prepare and test fragments and variants of a 2-phenylethanol dehydrogenase of the invention and determine whether the fragment or variant retains functional enzymatic activity relative to full-length or a wildtype plant 2-phenylethanol dehydrogenase. Similarly, an assay for the conversion of phenylalanine to phenethylamine can be used to assess enzymatic activity of fragments and/or variants of phenylalanine decarboxylase of the present invention.

Polynucleotides and polypeptides contemplated within the scope of the subject invention can also be defined in terms of more particular identity and/or similarity ranges with those sequences of the invention specifically exemplified herein. The sequence identity will typically be greater than 60%, preferably greater than 75%, more preferably greater than 80%, even more preferably greater than 90%, and can be greater than 95%. The identity and/or similarity of a sequence can be 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% as compared to a sequence exemplified herein. Unless otherwise specified, as used herein percent sequence identity and/or similarity of two sequences can be determined using the algorithm of Karlin and Altschul (1990), modified as in Karlin and Altschul (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990). BLAST searches can be performed with the NBLAST program, score=100, wordlength=12, to obtain sequences with the desired percent sequence identity. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al. (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (NBLAST and XBLAST) can be used. See NCBI/NIH website.

The subject invention also contemplates those polynucleotide molecules having sequences which are sufficiently homologous with the polynucleotide sequences exemplified herein so as to permit hybridization with that sequence under standard stringent conditions and standard methods (Maniatis et al., 1982). As used herein, “stringent” conditions for hybridization refers to conditions wherein hybridization is typically carried out overnight at 20-25 C below the melting temperature (Tm) of the DNA hybrid in 6×SSPE, 5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. The melting temperature, Tm, is described by the following formula (Beltz et al., 1983): Tm=81.5 C+16.6 Log [Na+]+0.41(% G+C)−0.61(% formamide)−600/length of duplex in base pairs.

Washes are typically carried out as follows:

(1) Twice at room temperature for 15 minutes in 1×SSPE, 0.1% SDS (low stringency wash).

(2) Once at Tm-20 C for 15 minutes in 0.2×SSPE, 0.1% SDS (moderate stringency wash).

As used herein, the terms “nucleic acid” and “polynucleotide” refer to a deoxyribonucleotide, ribonucleotide, or a mixed deoxyribonucleotide and ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, would encompass known analogs of natural nucleotides that can function in a similar manner as naturally-occurring nucleotides. The polynucleotide sequences include the DNA strand sequence that is transcribed into RNA and the strand sequence that is complementary to the DNA strand that is transcribed. The polynucleotide sequences also include both full-length sequences as well as shorter sequences derived from the full-length sequences. Allelic variations of the exemplified sequences also fall within the scope of the subject invention. The polynucleotide sequence includes both the sense and antisense strands either as individual strands or in the duplex.

The subject invention also concerns cells transformed with a polynucleotide of the present invention encoding a 2-phenylethanol dehydrogenase of the invention. In one embodiment, the cell is transformed with a polynucleotide sequence comprising a sequence encoding the amino acid sequence shown in SEQ ID NO: 2, or an enzymatically active fragment or variant thereof. In a specific embodiment, the cell is transformed with a polynucleotide sequence shown in SEQ ID NO: 1, or a sequence encoding an enzymatically active fragment or variant of SEQ ID NO: 2. In one embodiment, the polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 2 comprises nucleotides 172 to 1155 of the nucleotide sequence shown in SEQ ID NO: 1. In another embodiment, a cell is transformed with a polynucleotide sequence comprising a sequence encoding the amino acid shown in SEQ ID NO: 11, or an enzymatically active fragment or variant thereof. In a specific embodiment, a plant is transformed with a polynucleotide sequence shown in SEQ ID NO: 10, or a sequence encoding an enzymatically active fragment or variant of SEQ ID NO: 11. In one embodiment, the polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 11 comprises nucleotides 1 to 990 of the nucleotide sequence shown in SEQ ID NO: 10.

The subject invention also concerns cells transformed with a polynucleotide of the present invention encoding a phenylalanine decarboxylase of the invention. In one embodiment, the cell is transformed with a polynucleotide sequence comprising a sequence encoding the amino acid sequence shown in SEQ ID NO: 5, 7, 9, or 13, or an enzymatically active fragment or variant thereof. In a specific embodiment, the cell is transformed with a polynucleotide sequence shown in SEQ ID NO: 4, 6, 8, or 12, or a sequence encoding an enzymatically active fragment or variant of SEQ ID NO: 5, 7, 9, or 13. In one embodiment, the polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 5 comprises nucleotides 1 to 1395 of the nucleotide sequence shown in SEQ ID NO: 4. In another embodiment, the polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 7, 9, or 13 comprises nucleotides 1 to 1413 of the nucleotide sequence shown in SEQ ID NO: 6, 8, or 12, respectively.

Preferably, the polynucleotide sequence is provided in an expression construct of the invention. The transformed cell can be a prokaryotic cell, for example, a bacterial cell such as E. coli or B. subtilis, or the transformed cell can be a eukaryotic cell, for example, a plant cell, including protoplasts, or an animal cell. Plant cells include, but are not limited to, dicotyledonous, monocotyledonous, and conifer cells. In one embodiment, the plant cell is a cell from tomato. In an exemplified embodiment, the plant cell is a cell from a petunia plant. Animal cells include human cells, mammalian cells, avian cells, and insect cells. Mammalian cells include, but are not limited to, COS, 3T3, and CHO cells.

Plants, plant tissues, and plant cells transformed with or bred to contain a polynucleotide of the invention are also contemplated by the present invention. Plants within the scope of the present invention include monocotyledonous plants, such as, for example, rice, wheat, barley, oats, sorghum, maize, sugarcane, pineapple, onion, bananas, coconut, lilies, grasses, and millet. Plants within the scope of the present invention also include dicotyledonous plants, such as, for example, tomato, peas, alfalfa, melon, chickpea, chicory, clover, kale, lentil, soybean, tobacco, potato, sweet potato, radish, cabbage, rape, grape, sunflower, lettuce, cucumber, watermelon, apple, citrus (e.g., orange, lemon, tangerine, grapefruit, lime), pear, plum, peach, fig, currant, muskmelon, squash, cherry, sugar beet, tea, strawberry, blackberry, blueberry, raspberry, loganberry, rose, chrysanthemum, sweet pepper, eggplant, and cotton; and conifers. Preferably, the plant, plant tissue, or plant cell is tomato. Ornamental and herb plants containing a polynucleotide of the invention are also contemplated within the scope of the invention. Ornamental plants include roses, petunias, carnations, orchids, tulips, gardenias, and the like. Herb plants include parsley, sage, rosemary, thyme, and the like. Techniques for transforming plant cells with a gene are known in the art and include, for example, Agrobacterium infection, biolistic methods, electroporation, calcium chloride treatment, etc. Transformed cells can be selected, redifferentiated, and grown into plants using standard methods known in the art. Thus, the subject invention also concerns transgenic plants, and tissue and cells thereof, that have a polynucleotide of the invention incorporated into their genome. The seeds and progeny of any transformed or transgenic plant cells or plants of the invention are also included within the scope of the present invention.

The subject invention also concerns methods for providing a plant with increased flavor or fragrance of fruit or flower by incorporating one or more polynucleotide of the present invention in the genome of the plant cells and expressing the polypeptide encoded by the polynucleotide. In one embodiment, a plant is grown from a transformed plant cell of the invention. Preferably, the polynucleotide encodes a 2-phenylethanol dehydrogenase or a phenylalanine decarboxylase derived from the same plant species as the plant. In one embodiment, the plant is tomato. In another embodiment, the plant is a rose or other scented ornamental. In those embodiments, where increased flavor of fruit is desired, preferably the polynucleotide(s) of the invention is expressed in the fruit. In those embodiments where increased or enhanced fragrance of fruit or flower is desired, preferably the polynucleotide(s) of the invention is expressed in the fruit and/or flower. In a specific embodiment, a polynucleotide encoding an amino acid sequence shown in SEQ ID NO: 2, or an enzymatically active fragment or variant thereof, is incorporated into a plant genome. In one embodiment, the plant is a tomato plant. In an exemplified embodiment, the plant is a petunia plant. In a specific embodiment, the polynucleotide comprises a nucleotide sequence shown in SEQ ID NO: 1, or a sequence encoding an enzymatically active fragment or variant of SEQ ID NO: 2. In one embodiment, the polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 2 comprises nucleotides 172 to 1155 of the nucleotide sequence shown in SEQ ID NO: 1. In a specific embodiment, a polynucleotide encoding an amino acid sequence shown in SEQ ID NO: 11, or an enzymatically active fragment or variant thereof, is incorporated into a tomato plant genome. In a specific embodiment, the polynucleotide comprises a nucleotide sequence shown in SEQ ID NO: 10, or a sequence encoding an enzymatically active fragment or variant of SEQ ID NO: 11. In one embodiment, the polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 11 comprises nucleotides 1 to 990 of the nucleotide sequence shown in SEQ ID NO: 10. In another specific embodiment, a polynucleotide encoding an amino acid sequence shown in SEQ ID NO: 5, 7, 9, or 13, or an enzymatically active fragment or variant thereof, is incorporated into a tomato plant genome. In a specific embodiment, the polynucleotide comprises a nucleotide sequence shown in SEQ ID NO: 4, 6, 8, or 12, or a sequence encoding an enzymatically active fragment or variant of SEQ ID NO: 5, 7, 9, or 13. In one embodiment, the polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 5 comprises nucleotides 1 to 1395 of the nucleotide sequence shown in SEQ ID NO: 4. In another embodiment, the polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 7 or 9 comprises nucleotides 1 to 1413 of the nucleotide sequence shown in SEQ ID NO: 6 or 8. The level of expression of a polynucleotide of the invention can be manipulated using standard methods known in the art, including the use of promoters that provide for low, intermediate or high levels of expression.

The subject invention also concerns methods for producing 2-phenylethanol. In one embodiment, recombinantly produced 2-phenylethanol dehydrogenase of the invention can be used to enzymatically convert a suitable substrate, such as phenylacetaldehyde, into 2-phenylethanol. In another embodiment, a microorganism, such as yeast or E. coli, can be transformed with and express a polynucleotide encoding a plant 2-phenylethanol dehydrogenase enzyme of the invention and, optionally, one or more enzymes, such as phenylalanine decarboxylase and phenylethylamine oxidase, that through their enzymatic reactions result in a suitable substrate (e.g., phenylacetaldehyde) for 2-phenylethanol dehydrogenase to convert to 2-phenylethanol. Transformed microorganisms can be grown and polynucleotides expressed constitutively or induced, and 2-phenylethanol isolated from the microorganisms.

The subject invention also concerns methods for producing phenethylamine. In one embodiment, recombinantly produced phenylalanine decarboxylase of the invention can be used to enzymatically convert a suitable substrate, such as phenylalanine, into phenethylamine. In another embodiment, a microorganism, such as yeast or E. coli, can be transformed with and express a polynucleotide encoding a plant phenylalanine decarboxylase of the invention and, optionally, one or more enzymes, that through their enzymatic reactions result in a suitable substrate (e.g., phenylalanine) for phenylalanine decarboxylase to convert to phenethylamine. Transformed microorganisms can be grown and polynucleotides expressed constitutively or induced, and phenethylamine isolated from the microorganisms.

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

Materials and Methods

Plant Material.

Tomato (Lycopersicon esculentum Mill. cv. M82) and Lycopersicon pennellii introgression lines 8-2 and 8-2-1 (Eshed and Zamir, 1994) were grown in the greenhouse or field under standard conditions. Petunia plants were grown in a greenhouse under standard conditions.

Volatile Collection.

Volatiles were collected from approximately 100 g of chopped ripe tomato fruit as described by Schmelz et al. (2003). Petunia volatiles were collected from five flowers from each plant harvested at dusk. Volatiles were separated on an Agilent DB-5 column and analyzed on an Agilent 6890N gas chromatograph.

Microarrays.

Tomato cDNA microarrays were as described in Moore et al. (2002). Total RNAs were isolated as described earlier (Clardi et al., 2000). Arrays were hybridized with Cy3 or Cy5 labeled cDNAs from M82 and introgression line 8-2-1 fruit. Arrays were performed multiple times and with dyes reversed to ensure accuracy of the expression data.

2-Phenylethanol Dehydrogenase Expression in E. coli.

A full-length 2-phenylethanol dehydrogenase cDNA was cloned by 5′ RACE from tomato fruit cDNA using primer 5′-TCCTTGGCCCCACCAAGAGAAAGCAAGTGCTGCGT-3′ (SEQ ID NO: 3). Following sequence analysis the full-length cDNA was obtained by PCR. The coding region was cloned into vector pDEST15 containing a GST tag (Invitrogen) by recombination, and transformed into E. coli strain BL21-SI (Invitrogen) for inducible protein expression. Enzyme activity of crude E. coli extracts was determined by the method of Larroy et al. (2002) using phenylacetaldehyde, cinnamaldehyde or vanillin as a substrate.

Production of Transgenic Petunia Plants.

The full-length 2-phenylethanol dehydrogenase cDNA (SEQ ID NO: 1) was cloned in a vector under the control of the figwort mosaic virus 35S promoter (Richins et al., 1987) and followed by the Agrobacterium nopaline synthase (nos) 3′ terminator. The transgene was introduced into Petunia hybrida cv. Mitchell Diploid by the method of Wilkinson et al. (1997) with kanamycin resistance as a selectable marker.

In Vivo Phenylalanine Decarboxylase Activity Assays.

Tomato (M82 or introgression line 8-2-1) fruit pericarp disks were incubated with 1 μCi universally-labeled ¹⁴C-phenylalanine for 8 hr. Production of ¹⁴C—CO₂ was measured by incubating the pericarp disk in a sealed flask with a 2N KOH filter paper disk suspended above the pericarp disk, followed by scintillation counting. ¹⁴C-phenylalanine and ¹⁴C-phenylethylamine were extracted from the pericarp disk and separated using an AG-1 (OH—) column in series with a BioRex-70 column as described by Rontein et al. (2001). Production of ¹⁴C-phenylethylamine was confirmed by thin layer chromatography.

Phenylalanine Decarboxylase Expression in E. Coli.

The full-length aromatic amino acid decarboxylase cDNAs were cloned by sequencing putative clones from the TIGR database. Following sequence analysis the full-length coding sequence was obtained by PCR, and cloned into vector pENTR/D-TOPO. The coding region was then cloned into vector pDEST15 containing a GST tag (Invitrogen) by recombination, and transformed into E. coli strain BL21-AI (Invitrogen) for inducible protein expression. Production of the recombinant protein was confirmed by protein blotting with anti-GST antibodies. Enzyme activity was determined by growing E. coli strains expressing the aromatic amino acid decarboxylases in media containing 19.4 mM phenylalanine. Volatile compounds were extracted from the cultures using an equal volume of hexanes. Extracts were concentrated and analyzed by gas chromatography on an Agilent DB-5 column on an Agilent 6890N gas chromatograph. Identification of phenethylamine was confirmed by GC-mass spectrometry as described by Schmelz et al. (2001).

Southern Blotting.

DNA from L. esculentum M82 or 8-2-1 or L. pennellii leaves was digested with EcoRI, EcoRV, DraI, HaeIII or ScaI. Southern blotting was performed as described by Sambrook et al. (1989).

Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

Example 1 Volatiles Analysis

Volatiles analysis by GC on a DB5 column indicated high levels of phenylacetaldehyde and 2-phenylethanol in ripe fruit from L. pennellii introgression line (IL) 8-2-1. Levels of other tomato volatiles were similar to control M82 ripe fruit (FIG. 1). Levels of 2-phenylethanol were approximately 250× higher in 8-2-1 fruit than in M82 fruit. Phenylacetaldehyde levels in IL8-2-1 fruit were approximately 20× higher than in control fruit (FIG. 2). IL8-2-1 fruit had a distinct floral (rose-like) aroma consistent with the floral aromas of phenylacetaldehyde and 2-phenylethanol.

Example 2 Microarray Analysis

Microarrays containing approximately 3,000 tomato cDNAs indicated that an alcohol dehydrogenase gene (cLET2M9) was more highly expressed in IL8-2-1 fruit than in control M82 fruit; whereas, a related tomato alcohol dehydrogenase gene was not upregulated in IL8-2-1 fruit (Table 5).

TABLE 5 Microarray gene expression data for two alcohol dehydrogenase-like genes Microarray clone Ratio cLET2M9 +2.2 cLEG71F12 −2.35 RNAs extracted from M82 and introgression line 8-2-1 fruit were compared using cDNA microarrays. Positive values indicate higher RNA expression levels in introgression line 8-2-1 fruit; negative values indicate higher expression in M82 fruit.

Since clone cLET2M9 was only a partial cDNA, the full-length cDNA sequence for this clone was obtained by 5′ RACE. The full-length cDNA sequence of 2-phenylethanol dehydrogenase was then obtained by PCR, and confirmed by sequence analysis (FIG. 3A). The deduced amino acid sequence of the 2-phenylethanol dehydrogenase is shown in FIG. 3B (SEQ ID NO: 2). In plants, a substrate such as phenylacetaldehyde can be converted to 2-phenylethanol by 2-phenylethanol dehydrogenase (FIG. 4).

Example 3 Enzyme Activity

The 2-phenylethanol dehydrogenase coding region of the full-length cLET2M9 and the related cLEG71F12 were cloned in vector pDEST15 with a GST tag and transformed into E. coli BL21-SI cells for inducible expression. The production of recombinant protein in E. coli was determined by Western blotting with an anti-GST antibody. Alcohol dehydrogenase activities on phenylacetaldehyde and several related substrates were determined spectrophotometrically by the reduction in levels of NADPH and a decrease in OD₃₄₀ (FIG. 5). The highest level of activity was observed with phenylacetaldehyde as a substrate. Lower activity levels were also observed with cinnamaldehyde as a substrate, whereas negligible activity was seen with vanillin as a substrate. Protein from cLEG71F12 expressed in E. coli showed very little activity on the three substrates tested.

Example 4 Expression of 2-Phenylethanol Dehydrogenase in Transgenic Petunia

Full-length tomato 2-phenylethanol dehydrogenase cDNA was introduced into petunia (cv. Mitchell Diploid) under control of the constitutively expressed figwort mosaic virus promoter. Several transgenic petunia lines had high levels of expression of the tomato gene in flowers (data not shown). Wild-type petunia flowers emit relatively high levels of phenylacetaldehyde and lower levels of 2-phenylethanol. However, the transgenic petunia flowers expressing the polynucleotide encoding tomato 2-phenylethanol dehydrogenase have higher levels of 2-phenylethanol and lower levels of phenylacetaldehyde than wild-type flowers (FIG. 6). Levels of other petunia flower volatiles were similar to wild-type in the transgenic flowers. A range of phenylacetaldehyde and 2-phenylethanol levels were seen in the transgenic lines, however the majority of the lines had higher levels of 2-phenylethanol and lower levels of phenylacetaldehyde than wild-type flowers (FIG. 7). Overall, these data indicate that the introduction of the 2-phenylethanol dehydrogenase tomato transgene results in the conversion of phenylacetaldehyde to 2-phenylethanol in petunia flowers.

Example 5 Activity of 2-Phenylethanol Dehydrogenase on Various Substrates

Kin's for alcohol dehydrogenase activities of E. coli expressed LePEDH on phenylacetaldehyde and related substrates. The 2-phenylethanol dehydrogenase coding region of the full-length cLET2M9 was cloned in vector pDEST17 with a His tag and transformed into E. coli BL21-AI cells for inducible expression. Alcohol dehydrogenase activities on phenylacetaldehyde and several related substrates were determined. The highest level of activity was observed with phenylacetaldehyde as a substrate. Lower activity levels were also observed with cinnamaldehyde and benzaldehyde as substrates, whereas no detectable activity was seen with salicylaldehyde as a substrate.

TABLE 6 Substrate specificity of LePEDH Substrate Km (μM) Phenylacetaldehyde 42.3 Benzaldehyde 240.1 Cinnamaldehyde 428.9 NADPH 54.2

Example 6 In Vivo Aromatic Amino Acid Decarboxylase Activity

A pathway for the formation of 2-phenylethanol in plants is shown in FIG. 4. In the pathway, phenylalanine is converted to phenethylamine by phenylalanine decarboxylase, followed by conversion of phenethylamine to phenylacetaldehyde by an amine oxidase. Phenylacetaldehyde is then converted to 2-phenylethanol by a 2-phenylethanol dehydrogenase. To establish the first step in this pathway, tomato fruit pericarp disks were incubated with ¹⁴C-phenylalanine. The formation of ¹⁴C-phenethylamine and ¹⁴C—CO₂, the products of the decarboxylase reaction were then determined (Table 7). Higher levels of both phenethylamine and CO₂ were formed in the IL8-2-1 pericarp disks than in the control M82 pericarp disks (Table 7), and correlate with the higher levels of phenylacetaldehyde and 2-phenylethanol in IL8-2-1 tomato fruit. These results indicate that phenethylamine can be an intermediate in the pathway to phenylacetaldehdye and 2-phenylethanol.

TABLE 7 Phenylalanine decarboxylase activity of M82 and IL8-2-1 tomato pericarp disks fed ¹⁴C phenylalanine. Line nCi CO₂ nCi phenethylamine M82 0.96 ± 0.05 0.36 ± 0.02 8-2-1 5.16 ± 2.36 2.08 ± 0.39 Pericarp disks were fed 1 μCi universally labeled ¹⁴C-phenylalanine for 8 hours, and amounts of ¹⁴C—CO₂ and ¹⁴C-phenethylamine produced were determined.

Example 7 Identification of Putative Aromatic Amino Acid Decarboxylase Genes

Conversion of phenylalanine to phenethylamine would be catalyzed by a phenylalanine decarboxylase. Therefore, the tomato sequence databases were searched for cDNAs with similarity to other aromatic amino acid decarboxylases. Several clones of L. esculentum cDNA sequences were identified, although two were more similar to histidine decarboxylases than aromatic amino acid decarboxylases. The full-length coding sequence of each of these genes was obtained by PCR, and confirmed by sequence analysis.

Example 8 Enzyme Activity

The coding regions of the full-length aromatic amino acid decarboxylases were cloned in vector pDEST15 with a GST tag and transformed into E. coli BL21-SI cells for inducible expression. The production of recombinant proteins in E. coli was confirmed by Western blotting with an anti-GST antibody. E. coli cultures expressing the putative aromatic amino acid decarboxylases were grown in the presence of phenylalanine. Volatile compounds were extracted from these cultures and analyzed by gas chromatography. E. coli cultures expressing two putative aromatic amino acid decarboxylases (cLEC73K23 and cLEC75E21) produced a compound with the same retention time as phenethylamine, while control cultures did not (FIG. 13). E. coli cultures expressing three other putative amino acid decarboxylases did not produce phenethylamine. The presence of phenethylamine in the samples was confirmed by GC-MS. The nucleic acid coding sequences and amino acid sequences of the phenylalanine decarboxylases are shown in FIGS. 8A-8B (SEQ ID NO: 4 and SEQ ID NO: 5) and FIGS. 9A-9B (SEQ ID NO: 6 and SEQ ID NO: 7). The cLEC73K23 and cLEC75E21 cDNA sequences exhibit 79% identity to one another, whereas the amino acid sequences are 85% similar and 81% identical to each other.

Example 9 Southern Blotting

To determine if either of the genes with phenylalanine decarboxylase activity could be responsible for the altered levels of phenylalanine-derived volatiles in 8-2-1 fruit with the L. pennellii introgression on chromosome 8, Southern blots on M82, 8-2-1 and L. pennellii genomic DNA were performed using cLEC73K23 as a probe. Polymorphisms between M82 and L. pennellii were observed with EcoRI, EcoRV and ScaI (FIG. 10). With each of these enzymes the 8-2-1 restriction pattern was identical to L. pennellii, indicating that the cLEC73K23 gene in introgression line 8-2-1 came from the L. pennellii parent.

Example 10 Cloning of the L. pennellii cLEC73K23 Sequence

The nucleic acid coding sequence of the L. pennellii cLEC73K23 gene was obtained by PCR with primers from the 5′ and 3′ ends of the L. esculentum cLEC73K23. The correct 5′ and 3′ ends of the L. pennellii gene were then obtained by 5′ and 3′ RACE. The full-length coding sequence of this gene and the amino acid sequence are shown in FIGS. 11A-11B (SEQ ID NO: 8 and SEQ ID NO: 9). The L. pennellii and L. esculentum cLEC73K23 cDNA sequences are 95% identical, whereas the amino acid sequences are 98% similar and 97% identical. An amino acid alignment of the L. pennellii and L. esculentum cLEC73K23 sequences and the L. esculentum cLEC75E21 sequences are shown in FIG. 12.

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated with the scope of the invention without limitation thereto.

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We claim:
 1. A polynucleotide expression construct comprising: a polynucleotide sequence encoding a plant aromatic amino acid decarboxylase, or an enzymatically active fragment thereof, wherein said decarboxylase comprises the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 13, or an enzymatically active fragment thereof, or said decarboxylase has 70% or greater sequence identity with the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 13, and exhibits aromatic amino acid decarboxylase enzymatic activity, and wherein said expression construct comprises a heterologous promoter that is functional in a plant cell.
 2. A cell transformed with: a polynucleotide encoding a plant aromatic amino acid decarboxylase, or an enzymatically active fragment thereof, wherein said decarboxylase comprises the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 13, or an enzymatically active fragment thereof, or said decarboxylase has 70% or greater sequence identity with the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 13, and exhibits aromatic amino acid decarboxylase enzymatic activity.
 3. The cell according to claim 2, wherein said polynucleotide is provided in a polynucleotide expression construct.
 4. A plant, plant tissue, or plant cell transformed with or bred to contain: a polynucleotide encoding a plant aromatic amino acid decarboxylase, or an enzymatically active fragment thereof, wherein said decarboxylase comprises the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 13, or an enzymatically active fragment thereof, or said decarboxylase has 70% or greater sequence identity with the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 13, and exhibits aromatic amino acid decarboxylase enzymatic activity.
 5. A method for providing a plant with increased flavor or fragrance, said method comprising incorporating in said plant: a polynucleotide encoding a plant aromatic amino acid decarboxylase, or an enzymatically active fragment thereof, wherein said decarboxylase comprises the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 13, or an enzymatically active fragment thereof, or said decarboxylase has 70% or greater sequence identity with the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 13, and exhibits aromatic amino acid decarboxylase enzymatic activity; and expressing the polypeptide encoded by said polynucleotide.
 6. A transgenic plant, plant tissue, or plant cell, wherein said plant, plant tissue or plant cell comprises incorporated in the genome of said plant, plant tissue, or plant cell: a polynucleotide encoding a plant aromatic amino acid decarboxylase, or an enzymatically active fragment thereof, wherein said decarboxylase comprises the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 13, or an enzymatically active fragment thereof, or said decarboxylase has 70% or greater sequence identity with the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 13, and exhibits aromatic amino acid decarboxylase enzymatic activity.
 7. The expression construct according to claim 1, wherein said decarboxylase comprises the amino acid sequence shown in SEQ ID NO: 5, or an enzymatically active fragment thereof.
 8. The expression construct according to claim 1, wherein said decarboxylase comprises the amino acid sequence shown in SEQ ID NO: 7, or an enzymatically active fragment thereof.
 9. The expression construct according to claim 1, wherein said decarboxylase comprises the amino acid sequence shown in SEQ ID NO: 9, or an enzymatically active fragment thereof.
 10. The expression construct according to claim 1, wherein said decarboxylase comprises the amino acid sequence shown in SEQ ID NO: 13, or an enzymatically active fragment thereof.
 11. The expression construct according to claim 1, wherein said polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 12, or said polynucleotide comprises a nucleotide sequence that hybridizes under stringent hybridization conditions with the nucleotide sequence shown in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 12, or the complement thereof, and said polynucleotide encodes a polypeptide having aromatic amino acid decarboxylase enzymatic activity.
 12. The expression construct according to claim 1, wherein said polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:
 4. 13. The expression construct according to claim 1, wherein said polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:
 6. 14. The expression construct according to claim 1, wherein said polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:
 8. 15. The expression construct according to claim 1, wherein said polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:
 12. 16. The expression construct according to claim 1, wherein said expression construct further comprises one or more regulatory elements operably linked to said polynucleotide.
 17. The expression construct according to claim 16, wherein said regulatory element is one or more of a promoter, transcription termination sequence, translation termination sequence, enhancer, or a polyadenylation sequence.
 18. The expression construct according to claim 1, wherein said promoter is a seed-specific promoter, a tissue-specific promoter, a constitutive promoter, a developmentally-regulated promoter, or an inducible promoter.
 19. The expression construct according to claim 18, wherein said constitutive promoter is a CaMV promoter, ubiquitin promoter, actin promoter, or NOS promoter.
 20. The expression construct according to claim 18, wherein said tissue-specific promoter is a fruit-specific promoter.
 21. The expression construct according to claim 20, wherein said fruit-specific promoter is an E8 promoter, a hybrid E4/E8 promoter, an LeExp-1 promoter, or a polygalacturonase-β subunit promoter.
 22. The expression construct according to claim 18, wherein said tissue-specific promoter is a flower organ-specific promoter.
 23. The plant, plant tissue, or plant cell according to claim 4, wherein said polynucleotide is provided in a polynucleotide expression construct.
 24. The plant, plant tissue, or plant cell according to claim 4, wherein said decarboxylase comprises the amino acid sequence shown in SEQ ID NO: 5, or an enzymatically active fragment thereof.
 25. The plant, plant tissue, or plant cell according to claim 4, wherein said decarboxylase comprises the amino acid sequence shown in SEQ ID NO: 7, or an enzymatically active fragment thereof.
 26. The plant, plant tissue, or plant cell according to claim 4, wherein said decarboxylase comprises the amino acid sequence shown in SEQ ID NO: 9, or an enzymatically active fragment thereof.
 27. The plant, plant tissue, or plant cell according to claim 4, wherein said decarboxylase comprises the amino acid sequence shown in SEQ ID NO: 13, or an enzymatically active fragment thereof.
 28. The plant, plant tissue, or plant cell according to claim 4, wherein said plant is a tomato, pea, alfalfa, melon, chickpea, chicory, clover, kale, lentil, soybean, tobacco, potato, sweet potato, radish, cabbage, rape, grape, sunflower, lettuce, cucumber, watermelon, apple, citrus, orange, lemon, tangerine, grapefruit, lime, pear, plum, peach, fig, currant, muskmelon, squash, cherry, sugar beet, tea, strawberry, blackberry, blueberry, raspberry, loganberry, rose, chrysanthemum, sweet pepper, eggplant, cotton, rice, wheat, barley, oats, sorghum, maize, sugarcane, pineapple, onion, bananas, coconut, lilies, grasses, millet, rose, petunia, carnation, orchid, tulip, or gardenia.
 29. The method according to claim 5, wherein said polynucleotide is provided in a polynucleotide expression construct.
 30. The method according to claim 5, wherein said decarboxylase comprises the amino acid sequence shown in SEQ ID NO: 5, or an enzymatically active fragment thereof.
 31. The method according to claim 5, wherein said decarboxylase comprises the amino acid sequence shown in SEQ ID NO: 7, or an enzymatically active fragment thereof.
 32. The method according to claim 5, wherein said decarboxylase comprises the amino acid sequence shown in SEQ ID NO: 9, or an enzymatically active fragment thereof.
 33. The method according to claim 5, wherein said decarboxylase comprises the amino acid sequence shown in SEQ ID NO: 13, or an enzymatically active fragment thereof.
 34. The method according to claim 5, wherein said plant is a tomato, pea, alfalfa, melon, chickpea, chicory, clover, kale, lentil, soybean, tobacco, potato, sweet potato, radish, cabbage, rape, grape, sunflower, lettuce, cucumber, watermelon, apple, citrus, orange, lemon, tangerine, grapefruit, lime, pear, plum, peach, fig, currant, muskmelon, squash, cherry, sugar beet, tea, strawberry, blackberry, blueberry, raspberry, loganberry, rose, chrysanthemum, sweet pepper, eggplant, cotton, rice, wheat, barley, oats, sorghum, maize, sugarcane, pineapple, onion, bananas, coconut, lilies, grasses, millet, rose, petunia, carnation, orchid, tulip, or gardenia.
 35. The transgenic plant, plant tissue, or plant cell according to claim 6, wherein said polynucleotide is provided in a polynucleotide expression construct.
 36. The transgenic plant, plant tissue, or plant cell according to claim 6, wherein said decarboxylase comprises the amino acid sequence shown in SEQ ID NO: 5, or an enzymatically active fragment thereof.
 37. The transgenic plant, plant tissue, or plant cell according to claim 6, wherein said decarboxylase comprises the amino acid sequence shown in SEQ ID NO: 7, or an enzymatically active fragment thereof.
 38. The transgenic plant, plant tissue, or plant cell according to claim 6, wherein said decarboxylase comprises the amino acid sequence shown in SEQ ID NO: 9, or an enzymatically active fragment thereof.
 39. The transgenic plant, plant tissue, or plant cell according to claim 6, wherein said decarboxylase comprises the amino acid sequence shown in SEQ ID NO: 13, or an enzymatically active fragment thereof.
 40. The transgenic plant, plant tissue, or plant cell according to claim 6, wherein said plant is a tomato, pea, alfalfa, melon, chickpea, chicory, clover, kale, lentil, soybean, tobacco, potato, sweet potato, radish, cabbage, rape, grape, sunflower, lettuce, cucumber, watermelon, apple, citrus, orange, lemon, tangerine, grapefruit, lime, pear, plum, peach, fig, currant, muskmelon, squash, cherry, sugar beet, tea, strawberry, blackberry, blueberry, raspberry, loganberry, rose, chrysanthemum, sweet pepper, eggplant, cotton, rice, wheat, barley, oats, sorghum, maize, sugarcane, pineapple, onion, bananas, coconut, lilies, grasses, millet, rose, petunia, carnation, orchid, tulip, or gardenia.
 41. The expression construct according to claim 1, wherein said polynucleotide is complementary deoxyribonucleic acid (cDNA). 